Taking advantage of excellent specificity and sensitivity of the reaction of antigenic substances such as human chorionic gonadotropin, growth hormone, insulin and immunoglobulins to bind to their antibodies, numerous attempts have been made to measure such antigenic substances and their antibodies and already many immunochemical measuring processes are in practical use.
For instance there are available the following processes: immunodifusion methods, in which the antigen and the antibody are reacted together in agar gel; agglutination reactions and agglutination inhibition reactions, in which blood cells or fine particles of polystyrene latex are employed as carriers of an antigen or antibody; radioimmunoassay (RIA), in which radioisotopes are employed as a labeling agent; enzyme immunoassay (EIA), in which enzymes are used to label the antigen or the antibody; and fluorescence immunoassay, in which fluorescent substances are used to label the antigen or antibody.
Meanwhile, for the purpose of measuring low molecular weight substances such as steroids, thyroid hormones, physiologically active peptides or amines, whose antibodies are difficult to produce, a competitive protein binding assay which utilizes the reaction with a receptor protein or binding protein, i.e., protein which specifically binds to such low molecular weight substances, has been employed. Recently, however, these substances have come to be measured by the same processes as applied to the above-mentioned antigenic high molecular substances, because the antibodies of even these low molecular weight substances are now relatively easy to produce.
These processes have respective characters and are found widely applicable. Among others, RIA and EIA, which highly excel in the sensitivity of measurement and quantitative precision, are widely used, the substances measurable by them including a great variety of high molecular substances such as protein hormones, immunoglobulins, virus and low molecular weight ones such as peptides, steroids, amines, synthetic medicines.
The principle is common to RIA and EIA; use of a labeled antigen in which the antigen is labeled by a labeling agent or use of a labeled antibody in which the antibody is labeled by a labeling agent.
The process utilizing a labeled antigen is a so-called competitive process in which an unknown amount of unlabeled antigen and a given amount of labeled antigen are made to react competitively with a given amount of an antibody, whereby the activities of the labeling agent which is bound to an antibody or the labeled antigen which is not bound to it are measured. In the meantime, a dilution series of a reference substance, whose concentration is known, is prepared and in the same way described above the activity of the labeling agent in each dilution is measured. A standard curve obtained by plotting the measured activities is used to determine the amount of the substance to be measured. As a means to separate the labeled antigen which is bound to the antibody and the one which is not, an insolubilized antibody is employed.
In the process after reaction between an unknown amount of antigen and a given amount of labeled antibody the activities of the labeled antibody which is bound to the antigen or the labeled antibody which is not bound to it are measured. In the meantime, a dilution series of reference substance, whose concentration is known, is prepared and in the same way described above the activity of labeling agent in each dilution is measured. A standard curve obtained by plotting the measured activities is utilized for determining the amount of the substance to be measured. As a means to separate the labeled antibody which is bound to the antigen and the one which is not, an insolubilized antigen or an insolubilized antibody is employed.
The process employing the insolubilized antibody is a so-called sandwich process, which highly excels in the sensitivity of measurement. Its advantages are, however, detracted by the disadvantages that an antibody with fewer binding sites than an antigen is made insoluble for the purpose, the reactivity with the antigen to be measured decreases; and the process is not available for measurement of haptens.
By contrast the process employing the insolubilized antigen, which suffers less decrease in reactivity due to the antigen being rendered insoluble, can measure haptens but it is low in the sensitivity of measurement for reasons stated later.